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RESEA R C H Open Access
Improving therapeutic HPV peptide-based
vaccine potency by enhancing CD4+ T help and
dendritic cell activation
Chao-Yi Wu
1
, Archana Monie
1
, Xiaowu Pang
5
, Chien-Fu Hung
1,4
, T-C Wu
1,2,3,4*
Abstract
Background: Effective vaccination against human papillomavirus (HPV) represents an opportunity to control
cervical cancer. Peptide-based vaccines targeting HPV E6 and/or E7 antigens while safe, will most likely require
additional strategies to enhance the vaccine potency.
Methods: We tested the HPV-16 E7 peptide-based vaccine in combination with a strategy to enhance CD4+ T
help using a Pan HLA-DR epitope (PADRE) peptide and a strategy to enhance dendritic cell activation using the
toll-like receptor 3 ligand, poly(I:C).
Results: We observed that mice vaccinated with E7 peptide-based vaccine in combination with PADRE peptide
and poly(I:C) generated better E7-specific CD8
+
T cell immune responses as well as significantly improved
therapeutic anti-tumor effects against TC-1 tumors compared to E7 peptide-based vaccine with either PADRE
peptide or poly(I:C) alone. Furthermore, we found that intratumoral vaccination with the E7 peptide in conjunction
with PADRE peptide and poly(I:C) generates a significantly higher frequency of E7-specific CD8
+
T cells as well as
better survival compared to subcutaneous vaccination with the same regimen in treated mice.

Conclusions: The combination of PADRE peptide and poly(I:C) with antigenic peptide is capable of generating
potent antigen-specific CD8+ T cell immune responses and antitumor effects in vaccinated mice. Our study has
significant clinical implications for peptide-based vaccination.
Introduction
Cervical cancer is the 2
nd
leading cause of cancer deaths
in women worldwide. The primary etiological factor in
the development of cervical cancer is infection by
human papillomavirus (HPV) [1]. HPV is one of the
most common sexually transmitted diseases in the
world. It is now known that cervical cancer is a conse-
quence of persistent infection with high-risk type HPV
[1-5]. HPV infection is a necessary factor for the devel-
opment and maintenance of cervical cancer and thus,
effective vaccination against HPV represents an oppor-
tunity to control cervical cancer (for reviews see [6,7].
Peptide-based vaccination has emerged as a potentially
important strategy for the development of therapeutic
HPV vaccination as they are considered to be safe, easy
to produce, and stable [8,9]. The most important factor
in the designing of therapeutic vaccines is t he choice of
target antigen. In the case of HPV, the early viral proteins
such as E6 and E7 represent ideal target antigens since
they are consiste ntly expressed in a majority of cervical
cancers and its precursor lesions and are essential for
transformatio n [10]. The high-affinity H-2D
b
-restricted
E7-specific CTL epitope aa49-57 (RAHYNIVTF) has

been previously used in vaccination studies against HPV
16-transformed tumor cells [11]. These studies have
shown t hat vaccination with the E7 peptide-based
vaccine with incomplete Freud’ s adjuvant induced
E7-specific CD8+ T cell immune responses which
resulted in antitumor effects in a preclinical model [11].
This study suggests that with an appropriate strategy,
such as selecting an appropriate adjuvant, it is feasible to
enhance peptide-based vaccine potency. Thus, it is
important to continue to identify strategies t o enhance
* Correspondence:
1
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore,
Maryland, USA
Full list of author information is available at the end of the article
Wu et al. Journal of Biomedical Science 2010, 17:88
/>© 2010 Wu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License ( which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
peptide-based vaccine potency that may potentially be
suitable for clinical translation.
One strategy to enhance peptide-based vaccine
potencyistoinduceCD4+Thelpercellimmune
responses. CD4
+
T helper cells are known to play an
important role in the generation of CD8
+
T cell immune
responses as well as memory T cell responses (for

review see [12]). Thus, it is desirable to design an
immunization regimen that is capable of generating
antigen-specific CD4
+
T cells. P reviously, a Pan HLA-
DR epitope peptide (PADRE) has been described that is
capable of binding to different MHC class II molecules
with high-affinity [13]. PADRE peptides have been used
in conjunction with other forms of vaccines to enhance
vaccine potency in preclinic al models [13-15]. PAD RE
peptide s have also been used in clinical trials with mini-
mal toxicity [16,17].
Another strategy to enhance the peptide-based vaccine
potency is to activate dendritic cells via toll-like recep-
tors (TLR). DC activation is a prerequisite to T cell
priming and the generation of antigen-specific immune
responses. In the presence of “alert” signals such as TLR
ligands or inflammatory cytokines, DCs are stimulated
to mature and differentiate into potent activators of
antigen-specific T cells (for review, see [18]). Toll-like
receptor 3 (TLR3) recognizes viral double-stranded
RNA and its synthetic analog polyriboinosinic:polyribo-
cytidylic acid (poly(I:C)) and induce inflammatory cyto-
kines and dendritic cell activation (for review see [19]).
Poly(I:C ) has also been used in clinical trials and shown
to have minimal toxicity [ 20]. Thus, poly(I:C) can
potentially be used in combination with peptide-based
vaccines to activate DCs and thus enhance the antigen-
specific immune responses in humans.
In the current study, we explored the combination of

an E7 peptide-based vaccine with PADRE peptide and
poly(I:C) in the generation of E7-specific T cell immune
responses and therapeutic antitumor effects. We
observed that mice vaccinated with E7 peptide-based
vaccine in combination with PADRE peptide and poly(I:
C) generate significantly higher frequency of E7-specific
CD8
+
T cells as well as significant therapeutic anti-
tumor effects against TC-1 tumors. Furthermore, we
found that intratumoral vaccination with the E7 pep-
tide-based vaccine in combination with PADRE peptide
and poly(I:C) generates even higher frequency of E7-spe-
cific CD8
+
T cells as well as better survival compared to
subcutaneous vaccination in treated mice.
Materials and methods
Mice
Female C57BL/6 mic e (5-8 week s old) w ere purchased
from the National Cancer Institute (Frederick, MD) and
maintained under specific pathogen-free conditions in
the oncology animal facility of the Johns Hopkins Hos-
pital (Baltimore, MD). Animals were used in compliance
with institutional animal health care regulations, and all
procedures were performed according to the Johns Hop-
kins Institutional Care and Use Committee approved
protocols.
Cells and antibodies
TC-1 cells, which are an E7-e xpressing murine tumor

model, were obtained by co-transformation of primary
C57BL/6 mouse lung epithelial cells with HPV-16 E6
and E7 and an activated ras oncogene as previously
described [21]. They were maintained in RPMI medium
(Invitrogen, Carlsbad, CA, USA) supplemented with
2 nM glutamine, 1 mM sodium pyruvate, 20 mM
HEPES, 50 μM b-mercaptoethanol, 100 IUml
-1
penicillin,
100 μgml
-1
streptomycin and 10% f etal bovine serum
(FBS) (Gemini Bio-Products, Woodland, CA, USA). Anti-
mouse CD8a mAb (clone 53.6.7), CD4 (cloneGK1.5) and
IFN-g (BD Pharmingen, San Diego, CA, USA) were used
for intracellular cytokine analysis.
Peptide vaccination
Peptide vaccines were prepared by different mixtures of
HPV-16 E7 (aa 49-57) peptide (RAHYNIVTE, 20 μg),
PADRE peptide (AKFVAAWTLKAAA, 20 μg), E7(aa
49-57)-PADRE fusion peptide (RAHYNIVTEAKF-
VAAWTLKAAA, 20 ug) and polyriboinosinic:polyribo-
cytidylic acid (poly IC, 20 μg) (Sigma-Aldrich®, USA)
within 100 μl of PBS. C57BL/6 mice were immunized
either subcutaneously at the inguinal area or directly
into the TC-1 tumor mass every week for 2 continuous
weeks or longer.
Intracellular cytokine staining and flow cytometry analysis
Splenocytes were harvested from mice 1 week after their
second vaccination. Prior to intracellular cytokine stain-

ing, 5×10
6
poo led splenocytes were incubated overnight
with 1 μgml
-1
E7 peptide (aa 49-57) or PADRE peptide
(AKFVAAWTLKAAA) in the presence of GolgiPlug
(BD Pharmingen, San Diego, CA) (1 μgml
-1
). The sti-
mulated splenocytes were then washed once with FACS-
can buffer and stained with phycoerythrin-conjugated
monoclonal rat anti-mouse CD8a or CD4. Cells were
subjected to intracellular cytokine staining using the
Cytofix/Cytoperm kit according to the manufacturer’ s
instructions (BD Pharmingen). Intracellular IFN-g was
stained with fluoresce in isothiocyanate-conjugated rat
anti-mouse IFN-g to identify the immune response and
cytokine levels. Flow cytometry analysis was per formed
using FACSCalibur with CELLQuest software (BD
Biosciences, MountainView, CA, USA).
Wu et al. Journal of Biomedical Science 2010, 17:88
/>Page 2 of 10
In vivo tumor treatment experiments
5-8-week-old C57BL/6 mice ( 5 per group) were chal-
lenged subcutaneously with 1×10
4
/mouse of TC-1
tumor cells. In general, mice injected with 10
4

TC-1
tumor cells will develop tumor in 100% cases. In addi-
tion, if left untreated, the tumor will eventually kill the
mice within 2 months. Three days after tumor chal-
lenge, the mice were immunized subcutaneously using
20 μg/mouse of HPV-16 E7 (aa 49-57) peptide, 20 μg/
mouseofPADREpeptideoramixtureofE7and
PADRE peptide (20 μg each) or the E7(aa49-57)-PADRE
fusion peptide (20 μg/mouse) with or without treatment
with 20 μg/mouse of poly(I:C). The mice were given
boosterwiththesamedoseeveryweekatthesamesite
until they died or the tumor reaches 2 cm in diameter.
Tumor growth was monitored twice a week by inspec-
tion and palpation.
Evaluation of tumor infiltrating lymphocytes
TC-1 tumors were harvested from euthanized mice after
the skin was disinfected and carefully dissected. Medium
(5 ml) was added and the tumor was disintegrated b y
tweezers by rubbing against the mesh thus releasing the
entrapped lymphocytes. Cells were then filtered and
treated with AKT lysing buffer (Qual ity Biological, INC.
MD, USA) before intracellular staining.
Statistical Analysis
All data expressed as means ± standard deviation (s.d.)
are representative of at least two different experiments.
Comparisons between individual data p oints for tumor
sizes were made using a Student’s t-test or repeated
measure ANOVA (analysis of variance) test. Differences
in survival between experimental groups were analyzed
using the log rank test. Tumor sizes were calculated

using the following equation: (tumor length × width ×
height)/2. Death of m ouse was arbitrarily defined as
tumor diameter greater than 2 cm.
Results
Mice vaccinated with the E7 peptide in combination with
PADRE peptide and poly(I:C) generate the highest
frequency of E7-specific CD8
+
T cells
In order to determine the antigen-specific T cell
immune responses in mice vaccinated with the combi-
nation of E7 peptide-based vaccine with PADRE peptide
and poly(I:C), we performed intracellular cytokine stain-
ing followed by flow cytometry analyses. C57BL/6 mice
(5 per group) were immunized subcutaneously with the
E7 and/or PADRE peptide-based vaccine with or with-
out poly(I:C) twice with a 1-week interval. One week
after the last vaccination, splenocytes from vaccinated
mice were harvested and characterized for E7-specific
CD8
+
T cells (Figure 1A and 1B) or PADRE-specific
CD4+ T cells (Figure 1C and 1D) using intracellular
IFN-g staining followed by flow cytometry analysis. As
observed in Figure 1A and 1B, we found that mice vac-
cinated with the E7 peptide in combination with
PADRE peptide and poly(I:C) generated a significantly
higher number of E7-specific IFN-g secreting CD8
+
T

cell s compared to mice vaccinated with E7 peptide with
PADRE alone or poly(I:C) alone (* p < 0.05). In addition,
we observed that mice vaccinated with PADRE peptide
with or without E7 peptide in combination with poly(I:
C) also generated significant increase in the n umber of
PADRE-specific CD4+ T cells compared to mice vacci-
nated without poly(I:C) (Figure 1C and 1D). Further-
more, mice vaccinated with E7 peptide in combination
with PADRE peptide and poly(I:C) generated the highest
number of E7-specific IFN-g secreting CD8
+
T cell
immune response among all the vaccination groups.
Thus, o ur data indicates that vaccination with the mix-
ture of E7 and PADRE peptide in combination with
poly(I:C) are capable of generating the best E7 peptide-
specific T cell immune responses in vaccinated mice.
We further compared the antigen-specific T cell
immune responses generated by vaccination with the
mixture of E7 and PADRE pept ide with poly(I:C) and
the E7-PADRE fus ion peptide with poly (I:C). Our data
indicate that the mixture of E7 and PADRE peptide gen-
erates significantly better E7-specific CD8+ T cell
immune responses compared to the E7-PADRE fusion
peptide (Figure 2A). In comparison, the E7 and PADRE
mixture generates significantly lower PADRE-specific
CD4+ T cell immune responses compared to the E7-
PADRE fusion peptide (Figure 2B).
We also characterized the long-term memory immune
responses ge nerated by vaccination with the mixture of

E7 and PADRE peptide and the E7-PADRE fusion pep-
tide. We found that there was no significant difference
in the E7-specific CD8+ T cell immune responses and
the PADRE-specific CD4+ T cell immune responses
generate d by the mixture of E7 and PADRE peptide and
the E 7+PADRE fusion peptide (Figure 2C and 2D).
Taken together, our data indicates that vaccination with
the mixture of E7 peptid e with PADRE peptide in com-
bination with poly(I:C) leads t o significantly higher p ep-
tide-specific immune responses compared to vaccination
with the E7-peptide fusion peptide.
Treatment with the E7 peptide in combination with
PADRE peptide and with poly(I:C) leads to better survival
in TC-1 tumor-bearing mice
In order to determine if tumor-bearing mice treated
with E7 peptide in combination with PADRE peptide
and poly(I:C) can demonstrate therapeutic antitumor
effects, we performed in vivo tumor treatment experi-
ments. C57BL/6 mice (5 per group) were challenged
Wu et al. Journal of Biomedical Science 2010, 17:88
/>Page 3 of 10
subcutaneously with TC-1 tumor cells in the right leg.
Three days later, mice were immunized subcutaneously
with the HPV-16 E7 (aa 49-57) p eptide with PADRE
peptide or with poly(I:C) or with both poly(I:C) and
PADRE peptide at 1-week intervals. Tumor-bearing
mice treated with PBS or with poly(I:C) and PADRE
without E7 peptide were used as controls. Tumor
growth was monitored twice a week by inspection and
palpation. As shown in Figure 3, treatment with E7 pep-

tide in combination with PADRE peptide and poly(I:C)
demonstrated significantly better survival in TC-1
tumor-bearing mice compared to treatment with E7
peptide with PADRE alone or poly(I:C) alone (p < 0. 05).
Thus, our data indicate that treatment with the E7 pep-
tide in combination with PADRE peptide and poly(I:C)
leads to better survival in TC-1 tumor-bearing mice.
Figure 1 Characterization of the number of E7-specific CD8+ T cells and PADRE-specific CD4+ T cells in vaccinated mice. C57BL/6 mice
(5 per group) were immunized subcutaneously using 20 μg/mouse of HPV-16 E7 (aa 49-57) peptide, 20 μg/mouse of PADRE peptide or a
combination of the two with or without treatment with 20 μg/mouse of poly(I:C). Mice received a booster dose one week later. One week after
the last vaccination, splenocytes from vaccinated mice were harvested and stimulated with the E7 or PADRE peptide. Cells were characterized
for E7-specific CD8
+
T cells or PADRE-specific CD4+ T cells using intracellular IFN-g staining followed by flow cytometry analysis. Splenocytes
without peptide stimulation were used as negative control. (A) Representative data of intracellular cytokine staining followed by flow cytometry
analysis showing the number of E7-specific IFNg+ CD8+ T cells in the various groups (right upper quadrant). (B) Bar graph depicting the
numbers of E7-specific IFN-g-secreting CD8
+
T cells per 3 × 10
5
pooled splenocytes (mean ± s.d.). (C) Representative data of intracellular cytokine
staining followed by flow cytometry analysis showing the number of PADRE-specific IFNg+ CD4+ T cells in the various groups (right upper
quadrant). (D) Bar graph depicting the numbers of PADRE-specific IFN-g-secreting CD4
+
T cells per 3 × 10
5
pooled splenocytes (mean ± s.d.).
Data shown are representative of two experiments performed. * indicates p < 0.05.
Wu et al. Journal of Biomedical Science 2010, 17:88
/>Page 4 of 10

Intratumoral vaccination with the E7 peptide in
combination with PADRE peptide and poly(I:C) generates
significantly higher frequency of E7-specific CD8
+
T cells
compared to subcutaneous vaccination
In order to determine whether intratumoral vaccination
with the E7 peptide in combination with PADRE pep-
tide and poly(I:C) would generate enhanced E7-specifi c
CD8+ T cell immune responses, C57BL/6 mice (5 per
group) were challenged subcutaneously with TC-1
tumor cells in the right leg. Three days later, mice
were immunized subcutaneous ly or intratumo rally with
the E7 and PADRE peptide-based vaccine with poly(I:
C) twice with a 1-week interval. One week after the
last vaccination, splenocytes from vaccinated mice
were harvested characterized for E7-specific CD8
+
T cells using intracellular IFN-g sta ining followe d by
flow cytometry analysis. As shown in Figure 4, mice
vaccinated intratumorally with the E7 peptide in com-
bination with PADRE peptide and poly(I:C) generated
a significantly higher number of E7-specific IFN-g
secreting CD8
+
T cells in the s plenocytes compared to
mice vaccinated subcutaneously with the same vaccine
regimen (* p < 0.05).
We then isolated the tumor-i nfiltrating lymphocytes
from tumor-bearing mice vaccinated with E7 peptide in

combination with PADRE peptide and poly(I:C) either
intratumorally or subcutaneously and compared the
immune responses. We observed that mice vaccinated
intratumorally with E7 peptide in combination with
PADRE peptide and poly(I:C) generated a significantly
higher percentage of tumor-infiltrating CD8+ T cells
(Figure 5A) as well as E7-specific CD8
+
T cells (Figure
Figure 2 Comparison of the number of E7-specific CD8+ T cells and PADRE-specific CD4+ T cells in mice vaccinated with the E7 and
PADRE mixture with poly(I:C) versus the E7-PADRE fusion peptide with poly(I:C). C57BL/6 mice (5 per group) were immunized
subcutaneously using the mixture of E7 and PADRE peptide with poly(I:C) or the E7+PADRE fusion peptide with poly(I:C). Mice received a
booster dose one week later. One week (A & B) or 5 weeks (C & D) after the last vaccination, splenocytes from vaccinated mice were harvested
and stimulated with the E7 or PADRE peptide. Cells were characterized for E7-specific CD8
+
T cells or PADRE-specific CD4+ T cells using
intracellular IFN-g staining followed by flow cytometry analysis. Splenocytes without peptide stimulation were used as negative control. (A) Bar
graph depicting the numbers of E7-specific IFN-g-secreting CD8
+
T cells per 3 × 10
5
pooled splenocytes (mean ± s.d.). (B) Bar graph depicting
the numbers of PADRE-specific IFN-g-secreting CD4
+
T cells per 3 × 10
5
pooled splenocytes (mean ± s.d.). (C) Bar graph depicting the numbers
of memory E7-specific CD8
+
T cells per 3 × 10

5
pooled splenocytes (mean ± s.d.). (D) Bar graph depicting the numbers of memory PADRE-
specific CD4
+
T cells per 3 × 10
5
pooled splenocytes (mean ± s.d.). Data shown are representative of two experiments performed.
Wu et al. Journal of Biomedical Science 2010, 17:88
/>Page 5 of 10
5B and 5C) in the TILs compared to mice vaccinated
with the same regimen subcutaneously (* p < 0.05).
Taken together, our data indicates that intratumoral
vaccination with E7 peptide in co mbination with
PADRE peptide and poly(I:C) is capable of generating
better E7-speci fic CD8+ T cell immune responses com-
pared to subcutaneous vaccination.
Tumor-bearing mice treated intratumorally with E7
peptide in combination with PADRE peptide and poly(I:C)
demonstrate enhanced antitumor effects and prolonged
survival
In order to determine if tumor-bearing mice treated
intratumorally with E7 peptide in combination with
PADRE peptide and poly(I:C) can demonstrate
enhanced therapeutic antitumor effects compared to
subcutaneous treatment, we performed in vivo tumor
treatment experiments. C57BL/6 mice (5 per group)
were challenged subcutaneously with TC-1 tumor cells
in the right leg. Three days later, mice were immunized
either subcutaneously or intratumorally with the E7
peptide-based vaccine in combination with PADRE

peptide and poly(I:C) at 1-week intervals. As shown in
Figure 6A, mice vaccinated intratumorally with the E7
peptide in combination with PADRE peptide and poly
(I:C) demonstrated significantly better survival compared
to mice vaccinated subcutaneously.
In order to compare the survival in tumor-bearing
mice treated via intratumoral injection with the various
reagents, tumor-bearing mice were treated via intratu-
moral injection using HPV-16 E7 (aa 49-57) peptide
with PADRE peptide or with poly(I:C) or with both poly
(I:C) and PADRE peptide. Tumor-bearing mice treated
with PBS or with poly(I:C) and PADRE without E7 pe p-
tide were used as controls. The mice were given booster
with the same dose every 5 days at the same site until
they died or the t umor reaches 2 cm in diameter. The
survival of tumor-bearing mice was analyzed by Kaplan
& Meier analysis. As shown in Figure 6B, tumor-bearing
mice vaccinated intratumorally with the E7 peptide in
combination with PADRE peptide and poly(I:C) demon-
strated significantly better survival compared to mice
treated with E7 peptide with PADRE alone or E7
Figure 3 In vivo tumor treatment experiments. C57BL/6 mice (5
per group) were challenged subcutaneously with 1×10
4
/mouse of
TC-1 tumor cells. Three days later, the mice were immunized
subcutaneously using HPV-16 E7 (aa 49-57) peptide with PADRE
peptide or with poly(I:C) or with both poly(I:C) and PADRE peptide.
Tumor-bearing mice treated with PBS or with poly(I:C) and PADRE
without E7 peptide were used as controls. The mice were given

booster with the same dose every week at the same site until they
died or the tumor reaches 2 cm in diameter. The survival of tumor-
bearing mice was analyzed by Kaplan & Meier analysis. Linear graph
depicting survival of TC-1 tumor bearing mice treated with the
combination of E7 and PADRE peptide with or without poly(I:C) (p
< 0.05). Data shown are representative of two experiments
performed.
Figure 4 Flow cytometry analysis to determine the number of
E7-specific CD8+ T cells in the splenocytes of mice vaccinated
subcutaneously or intratumorally. The TC-1 tumor-bearing C57BL/
6 mice (5 per group) were immunized subcutaneously or
intratumorally using a combination of 20 μg/mouse of HPV-16 E7
(aa 49-57) peptide and 20 μg/mouse of PADRE peptide with 20 μg/
mouse of poly(I:C) twice with a 1-week interval. One week after the
last vaccination, splenocytes were harvested and characterized for
E7-specific CD8
+
T cells using intracellular IFN-g staining followed by
flow cytometry analysis. (A) Representative flow cytometry data
showing the number of E7-specific IFNg+ CD8+ T cells in
splenocytes from mice vaccinated subcutaneously or intratumorally
(right upper quadrant). (B) Bar graph depicting the numbers of E7-
specific IFN-g-secreting CD8
+
T cells per 3 × 10
4
pooled splenocytes
(mean ± s.d.). * indicates p < 0.05. Data shown are representative of
two experiments performed.
Wu et al. Journal of Biomedical Science 2010, 17:88

/>Page 6 of 10
peptide with poly(I:C) alone (p < 0.05). Taken together,
our data indicate that intratumoral vaccination with the
E7 peptide in combination with PADRE peptide and
poly(I:C) generates significantly enhanced therapeutic
anti-tumor effects against TC-1 tumors.
Discussion
In the current study, we observed that mice vaccinated
with the E7 peptide-based vaccine combined with
PADRE peptide and poly(I:C) generate the strongest E7-
specific CD8
+
T cell immune respon ses and therapeutic
anti-tumor effects against TC-1 tumors among the dif-
ferent vaccination groups. Furthermore, we found that
intratumora l vaccination with the E7 peptide-based vac-
cine in combination with PADRE peptide and poly(I:C)
generates significantly higher frequency of E7-specific
CD8
+
T cells as well as better survival compared to
subcutaneous vaccination with the same regimen in
treated mice.
We observed that the inclusion of PADRE peptide
could significantly improve the E7-specific immune
responses generated by the E7 peptide-based vaccine in
vaccinated mice. Our data is consistent with our pre-
vious studies using DNA-based vac cines. We have pre-
viously employed a DNA vaccine encoding an invariant
(Ii) chain in which the CLIP region is replaced with the

PADRE epitope (Ii-PADRE) [22]. We demonstrated that
mice vaccinated with DNA encoding Ii-PADRE showed
significantly greater PADRE-specific CD4
+
T cell
immune responses compared to mice vaccinated with
DNA encoding Ii chain alone [22]. More importantly,
co-administration of DNA encoding HPV E7 antigen
with Ii-PADRE D NA led to signific antly higher fre-
quency of E7-specific CD8
+
T cell immune responses
Figure 5 Flow cytometry analysis to determine the number of E7-specific CD8+ T cells in the tumor-infiltrating lymphocytes of mice
vaccinated subcutaneously or intratumorally. The TC-1 tumor-bearing C57BL/6 mice (5 per group) were immunized subcutaneously or
intratumorally using a combination of 20 μg/mouse of HPV-16 E7 (aa 49-57) peptide and 20 μg/mouse of PADRE peptide with 20 μg/mouse of
poly(I:C) twice with a 1-week interval. One week after the last vaccination, TILs were harvested and characterized for E7-specific CD8
+
T cells
using intracellular IFN-g staining followed by flow cytometry analysis. (A) Bar graph depicting the numbers of tumor-infiltrating CD8
+
T cells from
mice vaccinated either subcutaneously or intratumorally (mean ± s.d.). (B) Representative flow cytometry data showing the percentage of E7-
specific IFNg+ CD8+ T cells in tumor-infiltrating lymphocytes derived from mice vaccinated subcutaneously or intratumorally (right upper
quadrant). (C) Bar graph depicting the numbers of tumor-infiltrating E7-specific CD8
+
T cells from mice vaccinated either subcutaneously or
intratumorally (mean ± s.d.). Data shown are representative of two experiments performed. * indicates p < 0.05.
Wu et al. Journal of Biomedical Science 2010, 17:88
/>Page 7 of 10
and more potent protective and therapeutic antitumor

effects against TC-1 tumors in treated mice [22]. Thus,
the induction of CD4+ T help by employment of the
PADRE strategy may be used in combination with DNA
or peptide-based vaccination in order to enhance the
antigen-specific immune responses and antitumor
effects.
In our study, we found that intratumoral administra-
tion of the E7 peptide-based vaccines in conjunction
with poly(I:C) generated significantly greater E7-specific
immune responses and antitumor effects compared to
subcutaneous vaccination (See Figures 4, 5 and 6). A
potential mechanism for the observ ed effect may be
related to the fact that the E7 CTL peptide may directly
bind to MHC class I molecule of tumor cells, thus ren-
dering them more susceptible to direct killing by E7-
specific CD8+ T cells. This may also result in release of
E7 antigen from the apoptotic tumor cells which may be
taken up by antigen-presenting cells, resulting in further
presentation of E7 antigen to CD8+ T cells (so called
cross-priming mechanism). These mechanisms may
potentially contribute to the observed enhancement in
the E7-specific CD8+ T cell immune responses and anti-
tumor effects against E7-expressing tumors.
Another imp ortant mechanism f or the observed
enhancement i n immune responses and antitumor
effects by intratumoral administration of the peptide-
based vaccine with poly(I:C) may be related to the
alteration of the tumor microenviron ment. Poly(I:C) has
previously been shown to trigger the maturation of DCs
and promo te the production of inflammatory Th1 cyto-

kines such as IL-12, while suppressing Th2 cytokines,
such as IL-10 in vitro [23,24]. Thus, w e speculate that
intratumoral administration of peptide-based vaccines
with poly(I:C) may potentially generate an Th1 anti-
tumor i nflammatory response in the tumor microenvir-
onment, thus contributing to the destruction of the
tumor. Furthermore, the released tumor antigen, such as
E7 may potentially be taken up by antigen-presenting
cells, leading to further activation of tumor-specific CD8
+ T cells (cross-priming mechanism). Thus, intratu-
moral administration of poly(I:C) with the peptide-based
vaccine may alter the tumor microenvironment to
enhance the E7-specific immune responses as well as
Figure 6 In vivo treatment experiments comparing subc utaneous vaccination and intratumoral vaccination. (A) Kaplan-Meier graph
depicting the survival of TC-1 tumor bearing mice treated either intratumorally or subcutaneously with the combination of E7 and PADRE
peptide with poly(I:C). The TC-1 tumor-bearing C57BL/6 mice (5 per group) were immunized either subcutaneously or intratumorally using the
combination of 20 μg/mouse of HPV-16 E7 (aa 49-57) peptide and 20 μg/mouse of PADRE peptide with 20 μg/mouse of poly(I:C). The mice
were given one booster with the same peptide regimen and dose every week at the same site until they died or the tumor reaches 2 cm in
diameter and survival was analyzed by Kaplan & Meier analysis. (B) Kaplan-Meier graph depicting survival of TC-1 tumor bearing mice treated
intratumorally with the various combinations of reagents. Tumor-bearing C57BL/6 mice (5 per group) were treated via intratumoral injection
using 20 μg/mouse of HPV-16 E7 (aa 49-57) peptide with 20 μg/mouse of PADRE peptide or with 20 μg/mouse of poly(I:C) or with both poly(I:
C) and PADRE peptide. Tumor-bearing mice treated with PBS or with poly(I:C) and PADRE without E7 peptide were used as controls. The mice
were given booster with the same dose every 5 days at the same site until they died or the tumor reaches 2 cm in diameter and survival was
analyzed by Kaplan & Meier analysis. Data shown are representative of two experiments performed.
Wu et al. Journal of Biomedical Science 2010, 17:88
/>Page 8 of 10
antitumor effect s generated by the E7 pep tide-based
vaccine.
In summary, our study demonstrates that intratumoral
administration of an E7-peptide-based vaccine in combi-

nation with PADRE peptide and poly(I:C) leads to
enhanced antitumor effects in treated mice. The
employment of intratumoral administration of the pep-
tide-based vaccines in conjunction with PADRE peptide
and poly(I:C) can potentially be applied for advanced
cervical tumors which are not surgically resectable to
improve the clinical outcome. However, this approach is
restricted to a particular E7 pept ide. For future clinical
translation, we would require the employment of long
overlapping peptides to overcome the limitation of
MHC restriction and include more E7 CTL epitopes.
Recent studies employing peptide vaccination using an
overlapping set of long peptides comprising the
sequencesoftheHPV16E6andE7oncoproteinshave
been shown to demonstrate significant tumor-specific
immune responses [25-27]. Thus, the employment of
PADRE peptide and poly(I:C) may potentially be used in
comb ination with overlapping peptide-based vaccines to
enhance the antigen-spec ific immune responses and
antitumor effects for the control of HPV-associated
malignancies.
Acknowledgements
This work was supported by the American Cancer Society (C.F. Hung) and
National Cancer Institute SPORE in Cervical Cancer P50 CA098252, the 1 RO1
CA114425-01 and the 1 P20 CA144801 (T C. Wu).
Author details
1
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore,
Maryland, USA.
2

Department of Obstetrics and Gynecology, Johns Hopkins
Medical Institutions, Baltimore, Maryland, USA.
3
Department of Molecular
Microbiology and Immunology, Johns Hopkins Medical Institutions,
Baltimore, Maryland, USA.
4
Department of Oncology, Johns Hopkins Medical
Institutions, Baltimore, Maryland, USA.
5
Department of Oral Diagnostic
Service, Howard University, Washington DC, USA.
Authors’ contributions
CYW was involved in the execution of the project. AM was involved in the
interpretation of the data and writing the manuscript. XP participated in the
design of the study and the statistical analysis. CFH and TCW provided
overall supervision and guidance for the project. All authors read and
approved the manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 7 April 2009 Accepted: 22 November 2010
Published: 22 November 2010
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doi:10.1186/1423-0127-17-88
Cite this article as: Wu et al.: Improving therapeutic HPV peptide-based
vaccine potency by enhancing CD4+ T help and dendritic cell
activation. Journal of Biomedical Science 2010 17:88.
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